The transformation of a normal cell into a cancer cell is the result of an accumulation of genetic mutations or epigenetic changes that lead to aberrant gene expression. Understanding where and when these changes occur in any given tumor type is a primary focus of cancer research. Since cellular transformation is often unique for each patient, a diagnostic test that provides a customized genome-based profile of a tumor is very desirable and is likely be a standard in the future of oncological medicine. Genetic mutations in transformed cells are now very commonly profiled using next-generation sequencing (NGS). Incorporation of NGS technology into routine clinical diagnostics, however, will require overcoming a number of hurdles including streamlining of sample processing.
Random, unbiased fragmentation of DNA is necessary for NGS and is a key step in building any genomic library for sequencing. When NGS is used to determine genetic changes, the desired length of each DNA fragment in base pairs (bp) depends on the maximum possible read length of the sequencer. If fragments exceed the maximum length, they will be incompletely read. If they are too small, or degraded, then they will be excluded from the read. Therefore, consistent DNA fragmentation is required for quality NGS data. DNA shearing is also used in processes such as chromatin immunoprecipitation (ChIP), formaldehyde-assisted interrogation of regulatory elements (FAIRE), and DNA sequencing. For each of these techniques, it is important that DNA is sheared to a consistent size in the shortest time possible. Chromatin from cells that have been subjected to formaldehyde crosslinking (e.g., from the ChIP and FAIRE techniques), are particularly resistant to conventional DNA shearing techniques. Thus, the time required to shear formaldehyde cross-linked DNA makes it difficult to integrate this step into automated protocols with large sample sets.
Conventional methods for fragmentation of DNA include enzymatic digestion, sonication, nebulization, and hydrodynamic shearing. While all of these techniques are widely used, each has advantages and disadvantages. Enzymatic digestion using DNase I, MNase, or restriction enzymes is very efficient, but introduces an enzyme bias. Regions of transcriptionally silent, tightly packed (heterochromatic) DNA and DNA with high G-C content can be refractive to enzymatic digestion and many enzymes only create nicks in the DNA instead of cutting completely though both strands. The nebulization process shears solubilized DNA by forcing it through a pressurized nozzle (atomization). This method is fast, but requires large quantities of DNA and often results in a large distribution in the DNA fragment size and cross-contamination between samples. Hydrodynamic shearing involves forcing solubilized DNA through a mesh. It has the advantage of rapidly producing small DNA fragments of nearly uniform length. This method, however, is costly and the screen used for shearing is prone to clogging and cross-contamination between samples. Similar to enzymatic digestion, heterochromatic DNA or DNA with high G-C content are very difficult to shear, which creates a bias toward better shearing efficiency in euchromatic and A-T rich regions. Although NGS involves fragmentation of purified genomic DNA, ChIP requires fragmentation of DNA from the nuclei of intact, formaldehyde cross-linked cells. Formaldehyde crosslinks protein to DNA, and as a result cells are very rigid and particularly resistant to lysis. Therefore, DNA fragmentation for fixed samples such as ChIP or FFPE tissue is even more challenging. The summary is that current DNA fragmentation methods are a bottleneck for diagnostic assays such as NGS and ChIP, and that a substantial improvement in methods for DNA fragmentation would have a significant impact.
Presently, DNA fragmentation in academic laboratories is commonly performed using a probe-based or acoustic sonicator. Sonication uses uncontrolled cavitation to shear DNA. Conventional DNA sonicators range from a single probe to multi-sample acoustic water bath sonicators. The method typically produces inconsistent results and is time consuming, thereby limiting its utility. DNA extracted from cells or tissue must be optimized each time to ensure that fragmentation occurs to the desired size range. In the case of formaldehyde cross-linked samples, checking DNA fragment size involves an overnight incubation, so optimization can take several days for each sample type. Therefore, an inexpensive method that provides shearing consistency independent of cell or tissue type and that can be performed rapidly would be very valuable and would have a significant impact on the use of technologies like NGS as a companion diagnostic for cancer.
There are few methods currently available to increase DNA fragmentation efficiency by an acoustic or probe sonicator. Borosilicate glass beads are sometimes added to the DNA mix during sonication, but these beads provide limited improvement and will reduce the lifetime of a probe sonicator by causing pitting in the probe. Another technique uses a vial that contains a rod that enucleates bubbles during sonication. Cavitation nucleated from this rod help to shear DNA. While these microbubble nucleating rods improve consistency results in the sonicator, they release plastic residue that can clog columns in downstream applications, and are very costly (five dollars per sample). Thus, current DNA fragmentation methods are a bottleneck for the creation of NGS libraries.
Accordingly, in light of these disadvantages associated with conventional techniques for DNA shearing, there exists a need for more consistent and efficient techniques for DNA shearing. More specifically, there exists a need for methods and systems for using encapsulated microbubbles to process biological samples.